Effects of organic solvents on immobilized enzyme catalyses. Chymotrypsin immobilized on Sephadex

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Abstract
The esterolytic activities of native chymotrypsin (CT) and immobilized CT-Sephadex were studied in the presence of up to 20% of the organic solvents methanol, ethanol, 2-propanol, tert-butyl alcohol, dioxane, or DMSO (dimethyl sulfoxide). The general cosolvent-induced inhibition of the native enzyme was attenuated for immobilized CT. Most noticeably, the apparent catalytic rate constants for the CT-Sephadex-catalyzed hydrolysis of N-acetyl-L-tyrosine ethyl ester were invariant over the 2-20% dioxane concentration range surveyed. In contrast, the activity of the native enzyme in 20 dioxane was only 3% the activity recorded in the absence of cosolvent. Increasing the hydrophobic character of the protic cosolvents destabilized the native enzyme but stabilized CT-Sephadex. Both native and immobilized CT displayed remarkable stability in 20% aqueous DMSO (t1/2 [half-life] > 200 days). At least part of the DMSO-induced inhibition of native CT and CT-Sephadex was offset by increasing the apparent pH of the reaction medium. The altered kinetic patterns for CT-Sephadex are best explained by the effects of diffusional limitations on the apparent enzyme activity. The best compromise solvent for preparative applications of CT-Sephadex was tert-butyl alcohol.